1. Field of the Invention
The present invention relates to compounds and reagents for enumerating cells in samples of whole blood and more particularly to quaternized derivatives of acridine orange and reagents incorporating such derivatives and their use in quantitatively determining reticulocyte levels in a whole blood specimen by fluorescence flow cytometry techniques.
2. Description of the Prior Art
In all the higher animals, blood consists of an aqueous fluid part (the plasma) in which are suspended corpuscles of various kinds: the red blood cells (erythrocytes), the white blood cells (leukocytes) and the blood platelets. Plasma has a composition comprising roughly 90% water, 9% protein, 0.9% salts and traces of other materials such as sugar, urea, uric acid and the like.
The cells or corpuscles of the peripheral blood (i.e. the blood outside the bone marrow) are divided into two main groups: the red blood cells (erythrocytes), whose primary object is to transport oxygen, and the white blood cells (leukocytes), whose primary functions relate to the immune system and the destruction of materials foreign to the body. In addition to these two main groups, the blood also contains the so-called blood platelets which are important in hemostatis.
The final stages of erythrocyte maturation occur after their release from the bone marrow while these cells are circulating in the peripheral blood. These young red cells, or "reticulocytes", have lost their nucleus and thus their ability to divide or to synthesize RNA. Although these functions have ceased, reticulocytes are still metabolically active and are capable of synthesizing protein, taking up iron for the synthesis of heme, and carrying out the necessary metabolic reactions required to maintain an energy rich state. These cells are usually distinguished from mature erythrocytes through the presence of the reticulum which give them their name. This reticulum may be dyed by such agents as brilliant cresyl blue, nile blue sulfate or new methylene blue after which quantitation of reticulocytes may be performed by way of manual observation under a microscope.
Although reticulocytes normally comprise about 0.5 to 2 percent of the total red blood cell population, this percentage can change dramatically under abnormal conditions. For example, reticulocyte counts have been used for many years as a diagnostic aid in studying blood dyscrasias and as an index of red cell regeneration following hemorrhage, as well as for monitoring early toxicity in chemotherapy of certain malignant diseases.
The use of fluorescing stains or dyes for analysis of blood cells has ben known for many years. Particularly, the utilization of acridine orange as a fluorescent dye to stain nucleic acid has been known for over forty years. S. Strugger, Fluorescence Microscope In Examination Of Bacteria In Soil. Canad J. Res. C 26: 188-193 (1948) J. B. Vander, et al., J. Lab. Clin. Med 62, 132 (1963) described the use of acridine orange for the identification of reticulocytes by fluorescence microscopy. However, this technique required visual examination of the sample and thus possessed the inherent disadvantages of such manual optical examination methods.
A quaternized acridine orange derivative and its interaction with DNA ar discussed in In Vitro Effects Of Acridine Interaction On RNA Polymerase Interactions With Supercoiled DNA, Robert S. Greene, et al., It. J. Biochem., Vol. 15, No. 10, pps. 1231-1239 (1983). A study as performed of the interactions of homologous E. coli RNA polymerase with a recombinant plasmid containing the origin sequence of the E. coli chromosome. These interactions were analyzed by perturbation of the DNA template, i.e. extracellular DNA, and its supercoiled conformation with the intercalating dye acridine orange and an N-10-benzyl derivative of acridine orange. Characterization of the drug mediated perturbations of the enzyme DNA interactions were accomplished by kinetic, electrophoretic and autoradiographic methods under conditions specific for RNA polymerase-template binding, initiation and transcription. Based on these studies, the authors concluded that acridine orange interfered with the RNA polymerase-DNA template interaction much more efficiently than the N-10-benzyl substituted acridine orange. In effect, the authors found that acridine orange was a more effective DNA intercalator than the acridine orange derivative. There is no discussion that the specific acridine orange derivative could be used as a dye for cell analysis using fluorescence flow cytometry or as an intercellular RNA marker in general.
Additionally, many different types of automatic apparatus have been disclosed for detecting and quantitating blood cells. Representative of such methods (some of which use acridine orange or other fluorescent dyes) are U.S. Pat. No(s). 3,497,690, 3,916,205, 3,864,171 and 4,027,971. While these references generally disclose the us of fluorescent dyes in a variety of apparatus, including a flow cytometer, they do not provide a method or composition for quantitating reticulocytes by fluorescence.
Of particular interest are Adams and Kamentsky U.S. Pat. No. 3,684,377 and Adams U.S. Pat. No. 3,883,247. These patents relate to method and dye compositions for quantitating cells (particularly white blood cells) using a metachromatic fluorochrome dye such as acridine orange.
The Adams and Kamentsky patent describes the use of a vital dye composition for differential blood analysis of living white cells which consist essentially of acridine orange having a concentration between 10.sup.-7 and 10.sup.-5 grams per ml, the acridine orange solution having a pH factor and an osmolality within the normal physiological ranges for human blood plasma. While the patent teaches that this composition is useful for identifying the various types of white blood cells and for distinguishing them from other bodies in the blood, there is no teaching that this composition has any use in the enumeration of reticulocytes.
The Adams patent represents a modification of the teaching of Adams and Kamentsky in that the white blood cells are treated under conditions in which the cells are "shocked" by exposure to a non-physiologic medium during staining. That is, the staining composition used in the Adams patent is made hypotonic, the osmolality thereof being generally below that normally found in human blood. The teaching of the Adams patent is that this hypotonic condition produces a differential rate of uptake of acridine orange dye by the various types of white blood cells, thus allowing them to be more clearly distinguished from one another than in previous techniques. Although the Adams patent does purport to disclose a method for the detection of reticulocytes, the method disclosed therein has been criticized as being practically useless for the quantitation of reticulocytes in Natale U.S. Pat. No. 4,336,029.
In contrast to the Adams and Kamentsky and the Adams patents in which the differentiation of the subtypes of white blood cells depends upon the rate of uptake of the acridine orange dye, the invention of the Natale patent depends upon removing the kinetic factors and increasing the degree of dye uptake so that the reticulocytes will absorb a maximum amount of acridine orange dye. With prior art staining reagents, the reticulocytes absorb only small amounts of dye and therefore yield only low levels of fluorescence in any fluorescence detecting method. These low levels of fluorescence could generally not be well detected over the background fluorescence and consequently only a portion of reticulocytes in the sample could be detected
The dye composition of the Natale patent consists essentially of an aqueous solution of the metachromatic fluorochrome dye acridine orange, a chelating agent (citrate), an amino-group reacting reagent, and (if needed) an buffer to maintain the final pH of this solution at approximately 7.4. The osmolality of the solution is maintained at approximately 0.26 osmolality units, the normal physiological level, either by the chelating agent or by addition of sodium chloride as required. The main purpose of the Natale teaching is to identify both reticulocytes and platelets simultaneously. In fact, the Natale reagent contains citrate ions to maximize platelet staining.
The Natale reagent contains very high concentrations of the acridine orange dye (10.sup.-2 gram per liter) which has been found to stain the fluid conduits within the instrument, including the flow cells, resulting in false positive readings for white cells. This staining also creates carryover problems requiring extensive washing of the system which adds time consuming steps to the assay protocol.
Furthermore, the prior art disclosures regarding acridine orange specifically taught against having maximum acridine orange uptake, since such maximum uptake would destroy the discrimination among the various subclasses of white blood cells which was the main object of these prior art methods. Therefore, the prior art Adams and Adams and Kamentsky patents cannot be said to teach the subject composition or method.
Accordingly, there exists a need for improved acridine orange dyes and reagents useful for the quantitation of reticulocytes by fluorescence flow cytometry techniques. It is a principal object of the present invention to provide such dyes and reagents.